Scientia Agricultura Sinica

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Scientia Agricultura Sinica. 2021, 54(6): 0-0.

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Breeding and Evaluation of Elite Rice Line ZY56

DongFeng QIU,PingJuan GE,Gang LIU,JinSong YANG,JianGuo CHEN,ZaiJun ZHANG

Scientia Agricultura Sinica. 2021, 54(6): 1081-1091. doi:10.3864/j.issn.0578-1752.2021.06.001

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【Objective】In order to avoid the influences of unknown genes, genetic background and other uncontrollable factors in breeding practice, experiments were conducted to create new high-quality rice germplasm using an improved method, which integrated high-throughput SNP genotyping with traditional breeding to improve breeding efficiency. 【Method】 Two varieties of rice (Jianzhen 2 and Ezhong 5) were used as parents to make a cross (Ezhong 5/Jianzhen 2), and Ezhong 5 was used as recurrent parent to obtain backcross generations, whose progenies were selected by pedigree method. Rice qualities were evaluated in generations from BC₁F₂ to BC₁F₄. An elite line, 4W1-056, was screened in the generation of BC₁F₇. DNA segments from Jianzhen 2, Ezhong 5 and 66 plants of 4W1-056 were PCR-amplified and sequenced using capture sequencing technologies, and 908 SNP sites were analyzed. Phylogenetic analyses were performed based on genotypes of SNP sites using p-distance method in software MEGA7. Novel lines were selected and evaluated based on agronomic traits and rice qualities in combination with the results from phylogenetic analyses. 【Result】An elite line 4W1-056 was obtained by means of crossing, backcrossing and selfing, which has desirable plant type, strong tillering ability, good lodging resistance for stocky stems, excellent appearance quality and preliminarily stable in agronomic traits. Sixty-six plants selected from 4W1-056 were clustered into three groups based on the results from phylogenetic analyses. The base substitution rate among cluster Ⅰ, Ⅱ and cluster Ⅲ was 0.1398, and that between cluster I and Ⅱ was 0.0662. The base substitution rate within any of these clusters was 0, which suggested that there were no genetic differences among individual plants within the same cluster. Cluster II was designated ZY56 as a new line based on its agronomic traits and rice qualities, and its appearance quality is better than that of 4W1-056, with a chalkiness score of 0.9, which is closer to the appearance quality performance of Ezhong 5. ZY56 and its original parents were detected using 8K RICE SNP chips, the results showed that 14.13% of ZY56’s chromosome segments were from Jianzhen 2 and 85.87% were from Ezhong 5, Chip detection further showed that most of the genes in ZY56 originated from Ezhong 5, and verified the selection results after crossing, backcrossing and selfing. The minimum effective accumulated temperature needed for basic vegetative growth in ZY56 was 760.5℃, the critical light length for reproductive growth was 14 h 13 min, and the effective accumulated temperature needed for panicle differentiation was above 711.5℃. The results of quality analyses from experiments at different sowing dates showed that ZY56 responded to light length significantly weaker than Ezhong 5, its photosensitivity was weak and growth was stable, which was conducive to the development of rice qualities, and the rice qualities of ZY56 were more stable than Ezhong 5. 【Conclusion】We proposed a new method for the selection in higher generations of a germplasm development program, in which high-throughput SNP genotyping technology was used to screen plants for genetic consistency, and eventually to find out the strains that conform to breeding targets. This method overcame the difficulty in the selection for agronomic traits by means of traditional pedigree in advanced generations, and circumvented the problems that similar types of strains were repeatedly selected and different types were missed. The method saved efforts of selection in higher generations, while increased the efficiency of selection, so it had value of popularization.

Analysis of Copy Number Variation of Glu-3 Locus in Common Wheat

Can CHEN,NanNan HAN,Yang LIU,XiaoWei SHI,HongQi SI,ChuanXi MA

Scientia Agricultura Sinica. 2021, 54(6): 1092-1103. doi:10.3864/j.issn.0578-1752.2021.06.002

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【Objective】The variations in numbers of copies of a gene is a common and important gene structure variation, which often effects individual phenotype. Low-molecular-weight glutenin subunit (LMW-GS) located at the Glu-3 loci, is an essential part of storage protein in wheat. As a hetero-hexaploid, the huge and complex wheat genome makes it difficult to detect numbers of gene copies by classical methods. A limited information is available about CNV analysis of LWM-GS. To screen reliable and stable internal reference genes and systems for complex genome and explore the CNV determination technology suitable in different wheat varieties, the techniques used for detection of gene copy numbers, based on droplet digital PCR (ddPCR) which improve the detection flux of target gene and determine the particular numbers of gene copies of Glu-3 loci. 【Method】In this paper, Acc1 was used as the internal reference gene. The internal reference primers and probes were designed by using the corresponding gene sequence. In order to analyze stability of numbers of copies of Acc1 in 12 common wheat varieties, we used qualitative and quantitative PCR. The genomic DNA of variety Gaoyou2018 with five different dilutions was used as template, and qRT-PCR was used to analyze the repeatability and accuracy of Acc1 internal reference system (primers and probes).The corresponding specific primers and probes were designed according to the LMW-GS gene sequence of Glu-A3 loci. Two different methods, qRT-PCR and ddPCR, were used to detect the numbers of copies of Glu-A3 loci in eight wheat varieties, in order to determine which method is more suitable for high-throughput detection of Glu-3 loci gene. The specific primers and probes were also designed according to the sequences of LMW-GS gene at Glu-B3 and Glu-D3 loci. The numbers of copies of LMW-GS gene at Glu-A3, Glu-B3 and Glu-D3 loci of 231 varieties were determined and analyzed by ddPCR. 【Result】The results showed that the gene copy numbers of Acc1 was consistent among varieties and different DNA concentrations of the same variety. The coefficient of variation (CV) between repeats was 0.07%-0.77%. It is also indicated from results that the Acc1 internal reference system constructed in this paper has good stability and repeatability. The results of qRT-PCR and ddPCR were consistent in detecting the numbers of copies of LMW-GS gene at Glu-A3 loci in 8 wheat varieties, which were 3, 5, 3, 4, 3, 3, 3 and 3, respectively. However, the CV among repeats detected by ddPCR was 0.30%-1.67%, which much lower than that by qRT-PCR. It showed that using ddPCR method to detect the gene copy numbers of Glu-3 loci gene is more stable and reliable. The numbers of copies of LMW-GS gene were 4 at Glu-A3, Glu-B3 and Glu-D3 having a frequency of 95%, 32.03% 28.57% respectively in 231 wheat varieties. The total variation range in numbers of copies of Glu-3 was 10-21, and the CV was 16.12%.【Conclusion】With good stability and repeatability, Acc1 could be used as internal reference gene for numbers of gene copies detection in wheat. Both qRT-PCR and ddPCR could be used in gene copy numbers detection with wheat genes, but the ddPCR is more simple, stable, reliable and has high detection flux.

Effect of rhcN Gene Mutation on Nodulation Ability of Soybean Rhizobium HH103

HanXi LIU,Hao LÜ,GuangYu GUO,DongXu LIU,Yan SHI,ZhiJun SUN,ZeXin ZHANG,YanJiao ZHANG,YingNan WEN,JieQi WANG,ChunYan LIU,QingShan CHEN,DaWei XIN,JinHui WANG

Scientia Agricultura Sinica. 2021, 54(6): 1104-1111. doi:10.3864/j.issn.0578-1752.2021.06.003

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【Background】 Soybean is an important economic crop, which is the main source of food and feed. Symbiosis is a special character of soybean to fix nitrogen in air and transferred into ammonia via rhizobia. Via symbiosis soybean can acquire enough nitrogen source for development. Rhizobial type Ⅲ effectors play pivotal roles in regulating the establishment of symbiosis. The rhcN gene codes an ATP transferase, which can regulate the secretion of type Ⅲ effector of rhizobium into host cells. 【Objective】To elucidate the symbiotic mechanism will help to improve the nitrogen fixation efficiency of soybean-rhizobium symbiosis system and is useful for friendly agricultural development. 【Method】In this study, rhcN mutant was constructed by tri-parental mating method and inserted a kanamycin gene in the downstream of rhcN start codon. The mutant was confirmed by PCR and Southern blot. To detect whether the expression of other type Ⅲ effectors was affected by rhcN mutant. The expression of NopC and NopT were identified by induced by genistein via Western blot method. Nodulation experiment were performed in Leonard jars, to detect the nodule phenotype of Williams82 after inoculation with the wild strain HH103 and derived HH103ΩrhcN mutant. Finally, one-hundred soybean germplasms with different genotypes were used for nodulation experiment. 【Result】The results of PCR and Southern blot confirmed that HH103ΩrhcN mutant was successful, and the extracellular protein identification supported that rhcN mutant can inhibit the secretion of type Ⅲ effectors NopC and NopT. The nodulation test of Williams82 showed that rhcN mutation can significantly inhibit the nodule number and dry weight. Using HH103ΩrhcN mutant and wild type HH103, 100 core germplasm accessions were identified, these results showed that rhcN mutation could reduce the root nodule phenotype of 80 soybean accessions, significantly increase the root nodule phenotype of 13 soybean accessions, and 7 soybean resources did not change significantly. 【Conclusion】The results showed that the abnormal secretion of type Ⅲ effector factors of rhizobium could affect the establishment of symbiotic system. rhcN played an important role in the formation of soybean rhizobium symbiosis system, and different soybean genetic background had different responses.

Advances in Cotton Growth and Development Modelling and Its Applications in China

TongYu HOU,TingLi HAO,HaiJiang WANG,Ze ZHANG,Xin LÜ

Scientia Agricultura Sinica. 2021, 54(6): 1112-1126. doi:10.3864/j.issn.0578-1752.2021.06.004

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Cotton is an important commodity crop, and its high-yield, high-quality and high-efficiency production have strategic significance for China. The cotton growth and development models (CGDM) simulate the dynamic interactions of physiological and structural processes with the environment, and predict the growth, yield and quality formation of cotton, to provide great assistance and convenience for the optimal decision of management practices. Based on the comprehensive introduction of technical principle, structural composition and functional characteristics of physiological model, structural model and functional-structural model of cotton, some further discussions were developed on the research and application of these models at domestic and abroad. Some further discussions were developed on the research and application of these models at domestic and abroad, particularly in the application of the CGDMs for the simulation of dynamic change of cotton growth and yield, for the optimization of irrigation, fertilization and pest management strategies, and for the regional-assessment of cotton production system in China. According to the actual situation of cotton production in China, combined with the development direction of agricultural informatization in our country, several suggestions were put forward for the research, development and application of cotton growth simulation model in terms of localization study, scale improvement and popularization demonstration, in order to further promote the modernization development of cotton industry in China.

Effects of Agronomic Managements on Yield and Lodging Resistance of Millet

CuiQing WU,JingXin SUN,PingYi GUO,HongFu WANG,XinHui WU

Scientia Agricultura Sinica. 2021, 54(6): 1127-1142. doi:10.3864/j.issn.0578-1752.2021.06.005

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【Objective】In order to study the effect of interaction treatment cultivation conditions on yield of millet and the biomechanical properties of quantitative evaluation of lodging resistance. 【Method】In this paper, using split zone design, four millet varieties with representativeness in production were selected. Based on interaction treatment cultivation conditions of different seeding density and fertilization, seeding density and water, water and fertilization conditions, the effects of cultivation measures on the growth and development of millet and the biomechanical properties of stem on lodging resistance were systematically studied. 【Result】(1) Except for the effect of water on the average diameter of millet stalk, the yield, photosynthetic physiological characteristics and stem characteristics of millet were significantly affected by four factors: millet variety, fertilization amount, sowing density and water content (P<0.001). (2) The 1 000-grain weight and yield of millet increased with the increase of fertilizer and water. With the increase of seed density, 1 000-grain weight decreased, and the yield increased first and then decreased. The SPAD value and the maximum fluorescence value (F_m) of millet flag leaf at flowering stage were significantly higher than that at filling stage. The SPAD value of flag leaf at flowering stage and F_m value of flag leaf at filling stage were used as the target traits of millet breeding. (3) The effect of sowing density on the lodging rate in millet was the greatest, followed by the influence of water and variety, while the effect of fertilization was the least. (4) The tensile force of the penultimate node of millet stem was negatively correlated with plant height, average length of nodes and moisture of stem nodes, and positively correlated with average diameter of nodes. 【Conclusion】Under the interaction treatment cultivation conditions of different agronomic measures, the single factor had the greatest influence on the yield of millet, plant photosynthetic physiological characteristics and stem mechanical properties. Selecting fine varieties, reasonable close planting (≤700 000 plants/hm²) and proper water and fertilizer were effective measures to improve the quality of millet, to increase yield, and to reduce the lodging rate in the field.

Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids

Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG

Scientia Agricultura Sinica. 2021, 54(6): 1143-1153. doi:10.3864/j.issn.0578-1752.2021.06.006

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【Objective】The objective of this study is to screen the dsRNAs with high efficiency and targeting degradation of tobacco mosaic virus (TMV), achieve its mass preparation, and to explore the mechanism. 【Method】Using CP, MP and RdRP genes encoded by TMV as target sequences, dsRNAs were synthesized in vitro and infiltrated into Nicotiana benthamiana. After 24 h, TMV was inoculated, total RNA and protein were extracted from samples 2 and 3 d after TMV inoculation, and the mRNA level and protein level of CP gene were used as indicators, combined with the biological symptoms of TMV, to comprehensively evaluate the inhibitory effect of each dsRNA on TMV. The fluorescence expression of TMV-30B on N. benthamianas and the hypersensitive necrosis reaction of TMV on Nicotiana tabacum var. Samsun NN were also combined. The dsRNA fragments that efficiently inhibit TMV were screened by comparing the dsRNAs corresponding to the six target sequences on the TMV genome. In order to obtain a large number of dsRNAs, the corresponding gene fragments of dsRNAs were inserted between the double T7 promoters of the prokaryotic expression vector L4440 and transformed into RNase III deficient Escherichia coli HT115 (DE3). And the siRNA generated after the prepared dsRNA spraying on tobacco was deeply sequenced to compare the effect of exogenous application of dsRNA on the small RNA expression characteristics and enrichment bands of TMV infestation. 【Result】RdRP1461-1774 dsRNAs with high effect on TMV CP gene expression were screened, and L4440-dsRdRP1461-1774 prokaryotic expression vector was constructed, which could induce the formation of target dsRNAs. The dsRNA extracted from bacteria solution could significantly inhibit TMV, when TMV-30B was applied to infect tobacco, the number of fluorescence decreased, the time of leaf wilting prolonged, and the number of necrosis spots decreased significantly. The results of small RNA sequencing showed that in the RNAi process induced by TMV infection, the sense and antisense chains produced siRNA with approximately equal frequency, while the infiltration of exogenous dsRNA resulted in the enrichment of siRNA in the target area, and the accumulation of antisense chain of siRNA increased sharply, the cumulative value of the corresponding sense chain decreased sharply. The application of exogenous dsRNA could change the abundance of siRNA expression. 【Conclusion】The targeted anti-TMV dsRNA sequences were screened by comparing the effects of dsRNA on the targeted anti-TMV infection in plants. Finally, a 313 bp long effective segment of RdRP gene was selected. This fragment of dsRNA can efficiently bind to the target gene and reduce the expression of TMV in infected plants. At the same time, a dsRNA prokaryotic expression system of RdRP1461-1774 gene was constructed to achieve low-cost and high-efficiency production, which provided the basis for the follow-up application of dsRNAs in the control of plant virus.

Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein

YuXin LIANG,JianXiang WU,XiaoYu LI,ChunYu ZHANG,JiChao HOU,XuePing ZHOU,YongZhi WANG

Scientia Agricultura Sinica. 2021, 54(6): 1154-1162. doi:10.3864/j.issn.0578-1752.2021.06.007

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【Objective】Potato virus Y (PVY) is one of the most serious viruses that affect the yield and quality of potato. At present, no effective virus treatment agent has been found, and application of virus-free seed potato is the main control measure to prevent PVY damage. The objective of this study is to establish a rapid detection method with high sensitivity and specificity, and to provide technical support for quality control of virus-free seed potato. 【Method】PVY coat protein (CP) was expressed as small polypeptides with overlapping parts, and the epitopes of PVY-CP were analyzed by Western blot. Matching experiments were performed on monoclonal antibodies that recognized different epitopes by conventional double antibody sandwich ELISA (DAS-ELISA), and a pair of monoclonal antibodies with the best detection effect was screened. At the same time, the captured and detected antibodies were confirmed. According to the standard of maximum P/N value, the working concentration of antibody was determined by square titration. The optimal co-incubation time of antibody and antigen was determined by control variable method. The sensitivity of rapid DAS-ELISA was tested with different concentrations of PVY-CP protein as antigen. Potato samples infected with different viruses were used as antigens to detect the specificity of rapid DAS-ELISA. Through rapid DAS-ELISA and RT-PCR detection of 50 samples of potato samples with suspected infection PVY collected in the field, the coincidence rate was tested. Finally, the established rapid DAS-ELISA was used to detect potato samples infected by different PVY strains. 【Result】A panel of monoclonal antibodies (9G6 and 3D3) that can be used for DAS-ELISA were screened using the expressed peptides of PVY CP, and a rapid DAS-ELISA was established based on this panel of MAbs. The ELISA plate coated with the capture antibody 9G6, and then co-incubated with detector antibody HRP labeled 3D3 (2 μg·mL^-1) and samples for 5 min at 37℃ after blocking the plate. The detection limit was 0.5 ng·mL^-1. The results of specific analysis showed that the established method was only positive for potato samples infected with PVY, and negative for potato virus S (PVS), potato virus M (PVM) and potato leaf-roll virus (PLRV). In comparison with RT-PCR, DAS-ELSIA had the coincidence rate of 96% through testing 50 potato simples, and the results of PVY^N and PVY^O potato samples were positive. 【Conclusion】 The established PVY detection method has high sensitivity and specificity, and can be completed within 30 min, which is convenient and fast, and provides key technical support for high-throughput detection of PVY and the production of virus-free seed potato.

Cloning of Heat Shock Protein Gene Ld-hsp70 in Leptinotarsa decemlineata and Its Expression Characteristics under Temperature Stress

HaiXia ZHENG,YuLin GAO,FangMei ZHANG,ChaoXia YANG,Jian JIANG,Xun ZHU,YunHui ZHANG,XiangRui LI

Scientia Agricultura Sinica. 2021, 54(6): 1163-1175. doi:10.3864/j.issn.0578-1752.2021.06.008

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【Objective】 Heat shock protein (HSP), a class of highly conserved proteins, is generally found in all the organisms, which plays an important role in response to stress resistance. In order to ensure the normal physiological activities of the organism, the expression of HSP can be generated rapidly under the adverse environmental conditions. The objective of this study is to clone heat shock protein 70 (HSP70) genes of the important quarantine pest Leptinotarsa decemlineata (Ld-hsp70s), analyze their expression characteristics under temperature stress, and to clarify the function of L. decemlineata HSP70 under temperature stress. 【Method】The cDNA sequences of HSP70 genes of L. decemlineata were obtained based on the NCBI database. The full length cDNAs encoding HSP70 genes of L. decemlineata were cloned by RT-PCR and RACE technology. The full-length sequences were spliced by DNAMAN software. The sequence characteristics of Ld-hsp70s were analyzed by bioinformatic methods. The phylogenetic tree with the homologous sequences of HSP70 from L. decemlineata and other insects was constructed using the neighbor-joining (NJ) method with MEGAX software. The expression profiles of Ld-hsp70s were analyzed by qRT-PCR. The specific primers were designed using Primer 5.0 software. 【Result】The cDNA sequences of three HSP70 genes of L. decemlineata were obtained from the NCBI database, and were named as HSP70a, HSP70b and HSP70c, respectively. Three full-length sequences were obtained by cloning and splicing, and were named as Ld-hsp70a, Ld-hsp70b and Ld-hsp70c, respectively. The GenBank accession numbers were KC544268, KC544269 and KC544270, respectively. Sequence analysis showed that complete conserved domain, three signature sequences of HSP70 family, signal sequences and motifs of subcellular location at the carboxyl (C)-terminal were found in amino acid sequences of the three Ld-hsp70s. The conserved EEVD motif in the C-terminal of Ld-hsp70a and Ld-hsp70c indicated that they were cytosolic HSP70. The conserved KDEL motif in the C-terminal of Ld-hsp70b indicated that it was endoplasmic reticulum HSP70. Phylogenetic tree analysis showed Ld-hsp70a and Ld-hsp70b were clustered together with HSP70 from other insect species, while Ld-hsp70c was clustered together with the reported HSP70 from L. decemlineata. Ld-hsp70a, Ld-hsp70b and Ld-hsp70c had highest homology with Propylaea japonica Pj-hsp70, Lissorhoptrus oryzophilus Lo-hsp70, and Colaphellus bowringi Cb-hsp70, respectively. qRT-PCR results showed that the expression of all three Ld-hsp70s could be induced by high and low temperatures. No significant difference was observed in the relative expression of the three Ld-hsp70s after exposure to different temperatures for 1 h. The relative expression level of Ld-hsp70a was significantly upregulated by 2.24 and 2.41 folds after exposure to -10℃ and 44℃ for 4 h in female and male of L. decemlineata, respectively. However, no significant difference was observed in the relative expression of Ld-hsp70b and Ld-hsp70c under temperature treatments for 4 h. Whether Ld-hsp70s in female or male, no significant difference was observed of different treatment times at the same temperature. 【Conclusion】Ld-hsp70a in L. decemlineata can respond to temperature stress and may play an important role in the adaptation to adverse temperatures. Ld-hsp70b and Ld-hsp70c are not very sensitive to temperature stress, suggesting functional differentiation of the three Ld-hsp70s in response to abiotic stress.

Effects of Straw Addition on Soil Organic Carbon and Related Factors Under Different Tillage Practices

BiSheng WANG,WeiShui YU,XuePing WU,LiLi GAO,Jing LI,XiaoJun SONG,ShengPing LI,JinJing LU,FengJun ZHENG,DianXiong CAI

Scientia Agricultura Sinica. 2021, 54(6): 1176-1187. doi:10.3864/j.issn.0578-1752.2021.06.009

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【Objective】Straw addition is an effective means to increase soil organic carbon, which is significant to ensure the sustainability of the organic carbon in the farmland system. This study aimed at investigating the effect of straw addition on soil organic carbon (SOC) and related factors under different tillage treatments, so as to provide a theoretical basis for the management of carbon sequestration and yield increase in northern dry farmland. 【Method】The field soil samples from long-term conventional tillage (CT) and no-tillage (NT) for in-lab incubation experiment were collected. Four treatments were set up, namely, conventional tillage soil without straw (CT), no tillage soil without straw (NT), conventional tillage soil with straw (CTS), and no-till soil with straw (NTS), respectively. Each treatment owned 15 repetitions. The incubation experiment was conducted in a constant temperature incubator at 25 ℃ for 180 days, and the soil samples were periodically taken to determine the content of SOC, aggregate composition, microbial biomass carbon and the activity of related enzymes. 【Result】(1) Straw addition significantly increased the content of soil organic carbon and large aggregates. Compared with CT, CTS increased SOC by 15%-46%; compared with NT, NTS increased SOC 12%-21%; compared to the initial organic carbon content, at the end of cultivation, CTS and NTS increased by 26.8% and 7.0%, respectively. CTS and NTS had the highest particle size of 2 000-250 μm, accounting for 41%-50% of all aggregates. Compared with CT, CTS increased the proportion of aggregates >250 μm by 235%-310%, and NTS increased the proportion of aggregates >250 μm by 96%-149%. (2) The addition of straw significantly increased the δ¹³C value of soil organic carbon. The CTS treatment was 80.93‰-115.22‰, NTS was 48.92‰-80.49‰; CTS straw-derived carbon was significantly higher than NTS by 13%-66%. (3) The addition of straw significantly increased the microbial biomass carbon (MBC) content, β-glucosidase (BG), β-cellobiosidase (CBH) and β-xylosidase (BXYL) activities. Compared with CT, CTS increased MBC content by 239%-623%, and increased BG, CBH and BXYL activity by 58%-170%, 52%-337% and 117%-170%, respectively; compared to NT, NTS increased MBC content by 124%-555%, and increased BG, CBH and BXYL activities by 28%-181%, 4%-304% and 13%-118%. (4) Soil organic carbon was significantly positively correlated with BG, CBH, BXYL activity, MBC and the proportion of >2 000 μm, 2 000-250 μm aggregates, and negatively correlated with the proportion of 250-53 μm and ＜53 μm aggregates. The activities of BG, CBH and BXYL showed a very significant positive correlation with each other, and were significantly positively correlated with MBC, >2 000 μm aggregates, 2 000-250 μm aggregates, and extremely negative with ＜53 μm aggregates. Linear correlation analysis results showed that water-stable macroaggregates (>250 μm) could explain 48% of organic carbon changes, MBC could explain 45% of organic carbon changes, and BG, CBH and BXYL enzyme activities could explain 66%, 44% and 53% of organic carbon changes, respectively. 【Conclusion】The addition of straw could significantly increase the content of soil organic carbon and macroaggregates, increase the number of microorganisms, and promote the soil enzyme activity. The impact on soil organic carbon and its related factors was greater in conventional tillage soils. In addition to the physical protection of aggregates, the sequestration of straw carbon in soil also depended on the role of microorganisms in the soil.

Characteristics of Microbial Biomass Phosphorus in Yellow Soil Under Long-Term Application of Phosphorus and Organic Fertilizer

YanLing LIU,Yu LI,Yan ZHANG,YaRong ZHANG,XingCheng HUANG,Meng ZHANG,WenAn ZHANG,TaiMing JIANG

Scientia Agricultura Sinica. 2021, 54(6): 1188-1198. doi:10.3864/j.issn.0578-1752.2021.06.010

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【Objective】 The seasonal dynamics of microbial biomass phosphorus (MBP) in yellow soils under different long-term fertilization treatments were studied to understand the influence of long-term application of P and organic fertilizer on P nutrients cycling and P nutrients available for plant uptake. 【Method】 Six treatments, including CK, NK, NPK, M, 0.5MNP and MNPK, were investigated in a 22-year (1995-2016) long-term fertilization experiment at Guiyang (Guizhou Province) of China. 0-20 cm soil layer was collected at before sowing, seedling stage, jointing stage, flowering stage and after harvest of maize, respectively, and to determine MBP, soil available P (AP), acid phosphatase (ACP) activity, and calculate the MBP turnover time and turnover amount. 【Result】 The results showed that, compared with NK treatment, the mean value of MBP content and microbial P content (MPC) in different maize growing periods under the treatments of P and organic fertilizer application were significantly increased by 123.9%-446.2% and 85.0%-268.2%, respectively, with MNPK treatment increasing the most. Under the same amount of P application, the MBP content under M and 0.5MNP treatments was 26.7% and 32.7% higher than that of NPK treatment. MBP and MPC of NK treatment were the lowest at flowering stage, while the MBP content of P and organic fertilizer application treatments were the highest in maize growth period. The MPC showed a "double peak" curve with the growth period, and the highest content was at seedling stage and flowering stage. Compared with CK, the MBP turnover time of NK treatment was shortened by 405 days, and the MBP turnover amount was increased by 50.9%. Compared with NK treatment, the MBP turnover amount increased by 19.2%-156.4%, and MBP turnover time was prolonged by 248-391 days under the treatments of P and organic fertilizer application, which indicated that application of P and organic fertilizer could increase MBP storage capacity and decrease its turnover rate. The amount of soil available P and P uptake of maize plant under the treatments of P and organic fertilizer application were 3.63-7.27 times and 1.77-1.97 times of NK treatment, respectively. The acid phosphatase activity in each treatment was ranked as NK>M, MNPK>CK, NPK, 0.5MNP, and the mean value of NK treatment increased by 10.2%-21.0% compared with other treatments. Correlation analysis showed that soil available P was the most important soil factor affecting the MBP content and MBP turnover amount, while acid phosphatase activity was the most important soil factor affecting the MBP turnover time. 【Conclusion】 To sum up, soil MBP could reflect the P supply level, and the storage and turnover of MBP were of great significance for improving the potential P supply capacity of yellow soil. Long-term application of P and organic fertilizer could increase soil available P sink, soil P cycling and P available for plant uptake, increase P uptake of maize plants.

Function Analysis of vvi-miR172s and Their Target Genes Response to Gibberellin Regulation of Grape Berry Development

XuXian XUAN,ZiLu SHENG,ZhenQiang XIE,YuQing HUANG,PeiJie GONG,Chuan ZHANG,Ting ZHENG,Chen WANG,JingGui FANG

Scientia Agricultura Sinica. 2021, 54(6): 1199-1217. doi:10.3864/j.issn.0578-1752.2021.06.011

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【Objective】 miR172 is an important regulator of plant growth and development. The purpose of this research was to clarify the roles and modes response to gibberellin (GA) of the miR172 family and its target genes in development of different tissues of grape berry. 【Method】The mature and precursor sequences of vvi-miR172a/b/c/d were cloned and identified by miR-RACE and PCR techniques from grapevine cv. Rosario Bianco. The target genes of vvi-miR172s were predicted by psRNA Target software, and the phylogenetic, structure analysis and subcellular localization were performed by bioinformatics tools. RLM-RACE verified the cleavage roles of four target genes by vvi-miR172s. The cis-elements analysis of their promoters was predicted by PLANTCARE software. The qRT-PCR method was used to detect the temporal and spatial expression patterns of vvi-miR172s and target genes in different tissues of grape berry induced by exogenous GA₃ application. 【Result】The mature and precursor sequences of vvi-miR172a/b/c/d were cloned, and four target genes (VvAP2, VvRAP2-1, VvRAP2-2 and VvRAP2-3) were identified from grape genome. The cleavage sites of vvi-miR172s on target genes were detected by RLM-RACE, which proved that VvAP2, VvRAP2-1, VvRAP2-2 and VvRAP2-3 were the true target genes. Gene structure analysis result showed that all target genes contained 10 exons, 9 introns and 2 AP2 domains. The type and number of motif elements were similar, indicating that gene structures were highly conserved. Phylogenetic analysis showed that VvAP2, VvRAP2-1, and VvRAP2-2 were closer to poplar, and VvRAP2-3 had high homology with soybean. The secondary structure prediction of target proteins indicated that they existed in the form of alpha-helix and further folded into the stable tertiary structure. Subcellular localization results showed that all target proteins were mainly located in the nucleus. Both vvi-miR172c and VvRAP2-1 had the hormone related cis-elements response to GA₃, suggesting that they might be involved in the regulation of grape berry development by responding to corresponding hormones. Microarray data analysis revealed that the expression patterns of vvi-miR172c and target genes were tissue or orGA₃n specific. RT-qPCR analysis showed that vvi-miR172a/b/d showed a decrease expression trend in pericarp, while VvRAR2-1 exhibited a reverse expression trend to the former, indicating that vvi-miR172a/b/d neGA₃tively regulated VvRAR2-1. However, in flesh, VvAP2 was contradictory to vvi-miR172d, with the increased expression during grape development, suggesting that there was a significant neGA₃tive correlation between vvi-miR172d and VvAP2. Interestingly, GA₃ treatment promoted the neGA₃tive regulation of vvi-miR172d on VvRAP2-1 and induced the neGA₃tive regulation of vvi-miR172c on VvAP2/VvRAP2-1/VvRAP2-3. 【Conclusion】VvAP2, VvRAP2-1, VvRAP2-2, and VvRAP2-3 were the real target genes of vvi-miR172s. Among the vvi-miR172 family, vvi-miR172 a/b/d might mediate VvRAP2-1 regulation of pericarp development, whereas vvi-miR172d might mediate VvAP2 involved in the development of grape flesh. vvi-miR172c/d and their target genes might participate in the regulation of grape pericarp and flesh development in response to exogenous GA.

Rescue and Molecular Marker Assisted-Selection of the Cold-Resistant Seedless Grape Hybrid Embryo

PeiPei ZHU,YiJia LUO,Wen XIANG,MingLei ZHANG,JianXia ZHANG

Scientia Agricultura Sinica. 2021, 54(6): 1218-1228. doi:10.3864/j.issn.0578-1752.2021.06.012

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【Background】The traditional methods of breeding seedless grape can only use the seeded variety as the female parent and the seedless variety as the male parent to cross, and the rate of the seedless hybrid is extremely low. As a new breeding technology, the embryo rescue can use seedless grapes as female parents, which expands the selection range of female parents, and greatly improves the efficiency of seedless grape breeding. However, the embryo development rate, germination rate and seedling formation rate in seedless grape embryo rescue are still low, which restricts the application of this technique. 【Objective】The main objective of this paper was to screen suitable female parents and embryo development medium for embryo rescue, and to create new cold-resistant seedless grape germplasm by embryo rescue and molecular marker assisted-selection, so as to provide a basis for improving the effect of seedless grape embryo rescue and for further breeding new cold-resistant seedless grape varieties. 【Method】Using the cold-resistant seedless grape cultivar Jupiter (Euro-American hybrid) and the seeded hybrid 00-1-5 ( V. vinifera × V. amurensis) as the male parents and five seedless grape cvs. (Ruby Seedless, Qinhong No.1, Qinhong No.2, Crimson Seedless and Qinxiu) as the female parents, respectively, the hybridization was carried out between seedless grape and cold-resistant grape. The immature fruits of each female parent at the suitable sampling time were collected for ovule culture in vitro. The solid-liquid biphasic MM3 and ER medium were used as the embryo development medium to compare the effects of different medium and female parents on the rate of embryo development, germination, normal seedlings and abnormal seedlings. WPM + 0.2 mg?L^-1 6-BA + 0.1 mg?L^-1 IAA solid medium was used as embryo germination and seedling formation medium. The embryo-rescue seedlings obtained after domestication in the greenhouse were transplanted to the field in spring. In addition, 2MS + 0.2 mg?L^-1 6-BA + 0.1 mg?L^-1 IAA medium was used for transforming deformed seedlings into normal seedlings, which was added with 0, 1.0 and 1.6 mg?L^-1 ZnSO₄, respectively. Finally, the molecular marker SCF27-2000 linked to the seedless gene was employed to detect the seedless hybrids for embryo-rescue seedlings. 【Result】For five cross combinations, 175 embryos from 2 158 ovules cultured in MM3 were obtained, which were further germinated and formed 118 hybrid plants. 74 embryos from 894 ovules cultured in ER medium were obtained and further germinated and formed 58 hybrid plants. Using 00-1-5 as male parent, seedless cv. Qinhong No.2 was much more suitable to be the female parent than Qinxiu, its embryo development rate and seedling rate were the highest, reaching 17.04 % and 7.41%, respectively. Using Jupiter as the male parent, Ruby Seedless × Jupiter had the best effect, with embryo development and seedling rate of 13.71% and 10.67%; the next was Qinhong No.1 × Jupiter, the embryo development rate and seedling rate were 14.39% and 4.71%, respectively. However, Crimson Seedless × Jupiter had the lowest embryo development rate and seedling rate, reached to 9.55% and 1.76%, respectively. The rates of embryo development and seedling formation of ovules inoculated into the MM3 medium were higher than that of the ER medium. The highest rate of deformed seedlings was Qinhong No.1 × Jupiter, while Ruby Seedless × Jupiter had no deformed seedlings. The rate of deformed seedlings in the ER medium was higher than that in the MM3 medium. The deformed seedlings of combination Qinhong No.2 × 00-1-5 were transformed to 2MS+1.6 mg?L^-1 ZnSO₄+0.2 mg?L^-1 6-BA+0.1 mg?L^-1 IAA, producing the best results with a conversion rate of 40.00%. Using the molecular marker SCF27-2000 linked to seedless character, 176 hybrid strains from 5 cross combinations were detected and there were 159 strains with the seedless molecular markers. 【Conclusion】 Ruby Seedless, Qinhong No.1 and Qinhong No.2 were suitable as the seedless female parents for embryo rescue in vitro, but Crimson Seedless and Qinxiu were not relevant. MM3 medium was more appropriate for embryo rescue in vitro than the ER medium. The 159 plants from embryo rescue with the molecular marker linked to the seedless gene were potential seedless hybrids.

Spatial Distribution of Phytic Acid and Minerals’ Availability in Pomelo Fruit

Biao SONG,KaiYue XU,XiaoHua WANG,JiuXin GUO,LiangQuan WU,Da SU

Scientia Agricultura Sinica. 2021, 54(6): 1229-1242. doi:10.3864/j.issn.0578-1752.2021.06.013

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【Objective】This study was aimed to elucidate the spatial distribution of phytic acid (PA), mineral concentration and their availabilities in pomelo fruits, and these findings could provide theoretical bases for biofortification of mineral nutrition and comprehensive nutritional evaluation of pomelo fruit. 【Method】Five representative pomelo cultivars (white-fleshed pomelo, golden-pomelo, red-fleshed pomelo, three-red pomelo, and red-albedo pomelo) grown in Pinghe County, Fujian Province, were used in the present study. At the ripening stage, the representative fruits from each cultivar were selected and divided the whole fruit into four spatial parts, i.e. flavedo, albedo, segment membrane and juice sac. The PA, mineral concentration and their availabilities were analyzed by iron precipitation spectrometry, inductively coupled plasma mass spectrometry (ICP-MS) and molar ratio of phytic acid to minerals in different spatial locations of pomelo fruit. In addition, Zn bioavailability was also evaluated by the ternary model, a mathematical model of zinc absorption in human intestine. 【Result】The concentration of phosphorus (total phosphorus) and inorganic phosphorus was highest in juice sac, but the concentration of PA was the lowest in juice sac. The PA concentration decreased persistently from the outer (flavedo) to the inner (pulp), i.e. flavedo > albedo > segment membrane > juice sac. The concentration of phytate-phosphorus in juice sac was only 4% of total phosphorus, while that in peel was 30%. In addition, the significant cultivar differences of PA were recorded in juice sac. The concentration of PA in juice sac of different pomelo cultivars was the highest in red-fleshed pomelo, while which was the lowest in three-red pomelo and white-fleshed pomelo, with 2.6-fold difference. However, non-significant difference was found in phosphorus and inorganic phosphorus among different pomelo cultivars. From the perspective of mineral distribution in pomelo, the higher concentration of calcium (Ca) was recorded in peel (flavedo, albedo, and segment membrane), while the higher concentration of phosphorus was found in juice sac. Iron (Fe) in peel was significantly higher than that in juice sac, and the variation was the largest in flavedo and juice sac. Influenced by both PA and minerals in different spatial location of pomelo fruit, [PA]/[Mg] and [PA]/[Fe] was the highest in flavedo, [PA]/[Zn] and [PA]/[Mn] was the highest in albedo, whereas [PA]/[Ca] was the highest in the juice sac. There were also significant cultivar differences in the mineral availability in juice sac. The [PA]/[Fe] of golden-pomelo was nearly six times higher than that of red-albedo pomelo. The [PA]/[Zn] of red-albedo pomelo was 3.6 times higher than that of white-fleshed pomelo and three-red pomelo. Generally, among the five pomelo cultivars, three-red pomelo and white-fleshed pomelo had the relatively low PA while high mineral availabilities. 【Conclusion】Significant cultivar and spatial positional differences existed in PA, minerals and their availabilities in pomelo fruits. Phosphorus in the pomelo juice sac mainly existed in the form of inorganic phosphorus, rather than PA. The results suggested that the inhibitory effect of PA on mineral availability was relatively small and limited in the juice sac. However, the concentration of PA in peel (flavedo, albedo) was relatively higher. Therefore, it is necessary to pay attention to the minerals availabilities during pomelo peel related deep food processing.

Evaluation of Rice Eating Quality and Optimization of Varieties of Southwest Indica Hybrid Rice Based on Three Taste Evaluation Methods

Hui LU,YuJie YUAN,SiQi ZHANG,Hong CHEN,Duo CHEN,XiaoYuan ZHONG,Bo LI,Fei DENG,Yong CHEN,GuiYong LI,WanJun REN

Scientia Agricultura Sinica. 2021, 54(6): 1243-1257. doi:10.3864/j.issn.0578-1752.2021.06.014

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【Objective】 Based on the three existing taste evaluation methods, the aim of this study was to research the eating quality characteristics of different indica hybrid rice varieties, and to select the excellent taste indica hybrid rice varieties in Southwest China. 【Method】 Based on the variety screening test in 2017, a single-factor randomized block field experiment was conducted in Yongsheng and Dayi in 2018. The eating quality was evaluated by two national standard sensory evaluation methods and taste analyzer for comparing the response of indica hybrid rice to sensory evaluation method, and the differences were compared among the 20 rice varieties. 【Result】 The eating quality of rice was regulated by multiple factors, the varieties with good taste had common characteristics, including excellent aroma, appearance, palatability, flavor and cold rice texture under the two sensory evaluations, as well as the appearance and taste value under the taste analyzer evaluation, and consequently to better eating quality. The path analysis showed that palatability had the highest contribution, followed by flavor and appearance, and the cold rice texture was the lowest. There were obvious differences in the eating quality of rice among different varieties. Under the three evaluation methods, Yixiangyou 2115, Nei 5 you 39, Fanyou 609, Huaxiangyou 1618, Chuanyou 6203 and Longliangyou 1146 all had excellent eating quality at 2 eco-points. Compared with Lüyou 4923 and Chuanyou 8377, Yixiangyou 2115 and Huaxiangyou 1618 had higher aroma, appearance, palatability, flavor, and consequently to higher eating quality. There were differences in the response of varieties to different evaluation method. Yuxiang 203 at Yongsheng and Y Liangyou 1 hao at Dayi belonged to poor taste varieties under 2 sensory evaluations, whereas both of them significantly higher than average under the evaluation of taste analyzer. The linear fitting results showed that the sensory hundred percentage point system (SHP) and the comprehensive grade rating (CGR) had a high linear fit, the correlation coefficient was r=0.94^***, the correlation coefficients of the taste analyzer value (TAV) to SHP and CGR were r=0.49 ^*** or r=0.53 ^***, respectively, and the degree of dispersion was too large to fully explain the internal relationship between each other. 【Conclusion】 The eating quality of rice was affected by ecological conditions and varieties. However, using a single taste evaluation method could not accurately evaluate the eating quality of rice varieties. Therefore, 6 varieties with good and stable taste, including Yixiangyou 2115, Nei 5 you 39, Fanyou 609, Huaxiangyou 1618, Chuanyou 6203 and Longliangyou1146, were selected by comprehensive used of the three current taste evaluation methods, which could be promoted as the high-quality taste varieties in Southwest China.

Influence of Exogenous Protein Addition on Whole Wheat Dough Properties and Bread Quality Characteristics

Di WU,JiaYu WANG,XiaoZhi TANG,QiuHui HU

Scientia Agricultura Sinica. 2021, 54(6): 1258-1269. doi:10.3864/j.issn.0578-1752.2021.06.015

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【Objective】The influence of two exogenous protein addition on thermomechanical properties, dynamic rheological properties and microstructure of whole wheat dough as well as quality characteristics of bread were investigated. The aim of the research was to provide a basis for further utilization of exogenous protein addition to ameliorate quality of whole wheat food and developing novel products of whole wheat food. 【Method】 Mixolab and dynamic rheometer were used to study the Influence of exogenous protein addition on thermomechanical and rheological properties of whole wheat dough, respectively. The dough microstructure was observed by scanning electron microscope (SEM). Meanwhile, whole wheat bread was prepared and tested to investigate the influence of exogenous protein addition on loaf and volume texture characteristic of bread. 【Result】 With the increasing egg white powder (EW) addition, the water absorption of whole wheat dough decreased, while dough development time and stability time increased. During heating process, peak viscosity of the mixed flour and setback value of starch increased. It was shown from dynamic rheological studies that with the increasing addition of EW, storage modulus and loss modulus of dough consistently decreased, while tan δ of dough increased. From SEM, the dough microstructure has been effectively improved, as the internal pore structure significantly reduced. Furthermore, more continuous and homogeneous dough structure well compensated for the structural damage caused by the bran in whole wheat dough. The specific volume of whole wheat bread increased as well as the bread crumb became loose and soft. The hardness and chewiness decreased, as there was no significant change on springiness, cohesiveness and resilience. Meanwhile, with the increasing sodium caseinate (SC) addition, the water absorption of whole wheat dough and degree of protein weakening increased, while dough development time and stability time first increased and then decreased. During heating process, peak viscosity of the mixed flour decreased while setback value of starch increased. It was shown from dynamic rheological studies that storage modulus and loss modulus of dough increased when SC addition was greater than 1%. From SEM, the dough microstructure gradually densified with the holes and crevices in dough gradually diminished. The specific volume of whole wheat bread first increased and then decreased as well as the bread crumb became relatively dense. It was shown from TPA that the hardness and chewiness gradually increased as well as the cohesiveness and resilience gradually decreased, as there was no significant change on springiness. 【Conclusion】The addition of EW could replace vital wheat gluten to some extent for effectively ameliorating the quality of whole wheat bread, whilst the utilization of SC should consider its higher water absorbing ability.

The Temperature-Humidity Index Estimated by the Changes of Surface Temperature of Broilers at Different Ages

YuYan YANG,YaoWen LI,Shuang XING,MinHong ZHANG,JingHai FENG

Scientia Agricultura Sinica. 2021, 54(6): 1270-1279. doi:10.3864/j.issn.0578-1752.2021.06.016

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【Objective】The present study was conducted to estimate the temperature-humidity index (THI) based on the variations of surface temperature (ST) of broilers raised at different relative humidity (RH) levels and increasing ambient temperature (AT). 【Method】At day of 28, 35, 42 and 49, thirty AA broilers were raised in two controlled climate chambers. The RH of two chambers was set at 50% and 80%, respectively, and the AT in two chambers was set at a same procedure with increasing gradually by one degree per 0.5 h from 18 ℃ to 33 ℃. The ST of broilers, as well as the AT in two chambers was recorded at 2 min intervals using mini temperature data loggers. The wet-bulb temperature of two chambers was recorded at 10 min intervals. The THI model as as follow: THI = a*T_dry-bulb + (1-a)* T_wet-bulb, the ‘a’ was weighting coefficient of T_dry-bulb, which was calculated when the coefficient of correlation between THI and ST reach to the maximum. 【Result】When the AT exceeded 24 ℃, the ST of broilers increased linearly with the AT and was affected by the RH. The present study estimated the THI for broilers at different ages by using the data when AT exceeded 24 ℃. The THI models for broilers at different age were as follow: THI_d28=0.82T_db+0.18T_wb; THI_d35=0.69T_db+0.31T_wb; THI_d42=0.67T_db+0.33T_wb; THI_d49=0.61 T_db+0.39T_wb. The linear correlation coefficients between THI and ST reached more than 0.96. In two independent experiments, it was verified again that there was a linear relationship between THI and ST, and the predicted ST by THI model was basically consistent with the actual measured results. 【Conclusion】The present results indicated that THI model had a good linear relationship with ST and was suitable for the evaluation of warm environment when the AT exceeded 24℃. The THI models for broilers at different ages were different, and the weighting coefficient of wet bulb temperature in THI models were increasing with the increase of broiler age.

Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process

JiaQi WANG,YuHong DONG,JuLing JIANG,JianNing QIAN,WenTao WEI,GuoLiang SONG,JinBo JIAO,XinXin GUAN,GuoBiao JI,YeXin ZHANG

Scientia Agricultura Sinica. 2021, 54(6): 1280-1287. doi:10.3864/j.issn.0578-1752.2021.06.017

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【Objective】Selected one suspension PK15 cell line which is suitable for PCV2 virus , and then developed the production process for PCV2 vaccines(source of virus, MOI and harvest time ), to provide basic theory and guarantee for large-scale production use suspension culture instead of adherent culture. 【Method】The PK15 primary cells were diluted using the limiting dilution method and seeded in 96-well plates. The cell growth and morphology were observed every 2 days. After 90% of the cells were overgrown, the cells were gradually expanded from 96-well plates to 24-well plates,to 12-well plates, to 6-well plate, and finally to the square flask, three PK15 clones with good morphology that can grow adherently were selected. Three PK15 clones with good morphology that can grow adherently were selected. Three clones (PK15-1C8, 2F11, 1E5) were directly seeding in PK15 serum-free medium at a density of 1×10⁶cells/mL and placed in a shaker incubator at 37 ℃, 5% carbon dioxide, and 120 r/min to continue culture. Monitor the cell density and viability every day, passage every 3 days, make the cells gradually adapt to the suspension environment, the PK15 clone can culture as a fully suspension cell line and can growth well in serum free media. After suspension cells stability passage and cell bank, compared the PCV2 virus content with three suspension clone cells, one clone cell was selected for PCV2 production. For different kinds of viruses (source from adherent cells or suspension cells), explore the infection MOI (0.1, 0.2, 0.5) and harvest time (48, 72, 96, 120h), determine the best production process of PCV2. 【Results】(1)The results showed that when the adherent cells passage to second generation in serum media CD PK15 259, the cells can suspended growth, continuous passage for eleven generation, suspension cells can growth stable, when seeding with 1×10⁶ /mL cells, cells can reach 10×10⁶ /mL cells when growth at 72h, viability rate is above 95%, and the doubling time is about 20h; (2) The three suspension cells use the same condition to infection PCV2 virus, the virus content of PK15-1C8 cloned cells can reach 10^6.4TCID₅₀/mL, clone PK15-2F11 (10^5.5TCID₅₀/mL), PK15-1E5 (10^5.6TCID₅₀/mL), the virus content of the three cloned cells was higher than that of the original clone (10^4.7TCID₅₀/mL), however, the virus content of PK15-1C8 cloned cells is higher and more stable, so it is determined as a cell for follow-up research; (3) After infecting PK15-1C8 cloned cells with seed virus (10^6.4TCID₅₀/mL) from adherent cells, the optimal process is 1×10⁶/mL cell density with 0.1MOI and harvest at 72h the virus content can reach 10^6.5TCID₅₀/mL. After infecting PK15-1C8 cloned cells with seed virus (10^6.3TCID₅₀/mL) from suspension cells, the optimal process is 1×10⁶/mL cell density with 0.2MOI and harvest at 72h the virus content can reach 10^7.3TCID₅₀/mL. 【Conclusion】Through the monoclonal screening of PK15 cells, adapted to suspension cells compared the PCV2 virus content of 3 suspension cells, a suspension cell with the highest virus content was determined, and based on this suspension cell, explored the process of PCV2 virus, and then established the PCV2 fully suspension, serum-free culture process. The SV15-1C8 cell proliferation PCV2 process of full suspension serum-free culture was established. This process used suspension cells to amplify PCV2 virus for seed poisoning for the first time. The highest virus content can reach 107.3TCID50/mL, which can be used for factory vaccine production. According selection sensitive clones and optimized the process for PCV2, can increase the virus titer, and realizes full suspension culture without serum, improved the production process of PCV2, improve the production efficiency, reduce the cost and improve the quality of the production. This process first use suspension cells to amplify the PCV2 virus. The highest virus content can reach 10^7.3TCID₅₀/mL, which can be used for large-scale PCV2 virus production.

Improvement of Nosema ceranae Genome Annotation Based on Nanopore Full-Length Transcriptome Data

HuaZhi CHEN,YuanChan FAN,HaiBin JIANG,Jie WANG,XiaoXue FAN,ZhiWei ZHU,Qi LONG,ZongBing CAI,YanZhen ZHENG,ZhongMin FU,GuoJun XU,DaFu CHEN,Rui GUO

Scientia Agricultura Sinica. 2021, 54(6): 1288-1300. doi:10.3864/j.issn.0578-1752.2021.06.018

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【Objective】The objective of this study is to improve gene sequence and functional annotation of current reference genome of Nosema ceranae using previously obtained Nanopore full-length transcriptome dataset. 【Method】TransDecoder software was used to predict open reading frames (ORFs) of N. ceranae and corresponding amid acids. Comparison between full-length transcripts and transcripts annotated in reference genome was performed using gffcompare software to extend upstream sequences or downstream sequences of annotated genes’ untranslated regions and correct genes’ boundaries. MISA software was used to explore simple sequence repeat (SSR) loci within transcripts with a length above 500 bp, including single nucleotide repeat, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat, pentanucleotide repeat, hexanucleotide repeat and mixed SSR. By using Blast tool, novel genes and novel transcripts were aligned to Nr, KOG, eggNOG, GO and KEGG databases to gain functional annotations. 【Result】A total of 2 353 complete ORFs were predicted, and those ORFs with a length distribution among 0-100 aa were the predominant, reaching a ratio of 72.12% among total ORFs. Additionally, structures of 2 340 N. ceranae genes were optimized; 5′ ends of 1 182 genes and 3′ ends of 1 158 genes were respectively prolonged. Moreover, 1 658 SSRs were identified, and the numbers of single nucleotide repeat, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat were 1 622, 23, seven and six, respectively. The density of single nucleotide repeat was the highest (182.32/Mb), followed by those of mixed SSR, dinucleotide repeat and trinucleotide repeat, reaching 6.90, 2.78 and 0.73/Mb, respectively. Further, 954 novel genes were identified, among them 951, 333, 371, 422 and 321 were respectively annotated to Nr, KOG, eggNOG, GO and KEGG databases. In addition, 6 164 novel transcripts were identified, among them 6 141, 2 808, 2 932, 3 196 and 2 585 were annotated to the aforementioned five databases, respectively. The species annotated by the highest number of new gene and new transcript was N. ceranae followed by Nosema apis. 【Conclusion】Our results well improve sequences and functional annotations of annotated genes in current reference genome of N. ceranae, and supplement and annotate a number of unannotated novel genes and transcripts. Lots of SSR sites were provided for research on molecular markers, information of genes and transcripts on reference genome were supplemented.

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